The immunoarchitecture of cutaneous pseudolymphoma

The Immunoarchitecture of Cutaneous Pseudolymphoma DANIEL P. WIRT, MD, THOMAS M. GROGAN, MD, CAROLYN S. JOLLEY, MT [ASCP], CATHERINE S. RANGEL, HTL [ASCP], CLAIRE M. PAYNE, PHD, RONALD C. HANSEN, MD, PETERJ. LYNCH, ME), AND MARIA SCHUCHARDT Fendt sarcoid, cutaneous lymphoplasia, lymphadenoid granttloma, lymphocytic infiltration of Jessner, lymphomatoid papulosis) reflects the morphologic diversity that can occur u n d e r this diagnostic heading, I-5 as well as the limitations o f rotttine morphologic analysis for tmderstanding the immunology and predicting the natural history of cutaneous lymphoproliferative disease. This historical circumstance is tmderstandable since routine morphologic observations alone cannot account for the dynamic immunologic processes that clearly underlie these pathologic conditions. To investigate the immunologic processes that underlie cutaneous i~seudolymphoma, we studied the i m m u n o a r c h i t e c t n r e of five cutaneous pseudolymphomas by staining serial frozen sections for T- and B-cell and dendritic reticuhmt cell (DRC) surface antigens with monoclolml antib6dies, allowing phenotypic analysis while maintaining architectural relatitmships. 6-8 Out- results demonstrate the validity and promise of this method for delineating the complex relationships of lymphoretict, lar cells in the skin, i.e., the immnnology of skin-associated lymphoid tissue (SAI:I'), and for distinguishing pseudolymphonaa from lymphoma in certain difficuh cases. From this study, c u t a n e o u s p s e u d o l y m p h o m a e m e r g e d as a reactive, hyperplastic proliferation of SALT. In some cases, B and T cells showed compartmental immnnoarchitectnre strikingly similar to that of reactive lymph nodes studied by the same method 6-8 (unpublished observations). In these cases the B- and T-cell compartments had their own populations of reticuhun cells, analogous in distribution and, probably, in function to the antigen-processing/presenting, lymphocyte-activating reticultml cells in the B- and T-cell c o m p a r t m e n t s of reactive lymph nodes. In some cases subtle immunologic evidence of aberrance, possibly identifying a subset at risk for malignant transformation, was observed. Although the main focus of this study was cases in which substantial I)opuhttions of both B and T cells were 1)resent, preliminary observations were m a d e for one case in which ,'I predominance of T cells and prominent epidermt)tropism simulated mycosis fimgoides.

The i m m u n o a r c h i t e c t u r e of five cutaneous p s e u d o l y m p h o m a s was studied by staining serial sections for T- and B-cell and dendritic reticulum cell (DRC) antigens with monoclonal antibodies, and compared with that of reactive lymph nodes and cutaneous lymphoma. In four cases compartmentalization of B and T cells was observed, analogous to findings in reactive lymph nodes. In two of these cases the immunoarchitectural features were strikingly sinfilar to those of reactive lymph nodes. Both had distinct follicles with germinal centers, and in one distinct mantle zone formation was seen. B cells in the follicles were polycional, with K chain p r e d o m i n a n c e . T h e g e r m i n a l centers showed the expected intercellular and/or dendritic pattern of immunoglobulin heavy chain, B2, and DRC-antigen expression. T cells admixed in the germinal centers were overwhelmingly of the T-helper type. T h e B-cell compartments in the other two cases showed some subtle immunologic evidence of aberrance, but the weight of evidence suggested reactive/aberrant rather than malignant processes. The T-cell compartments in all four cases showed a predominance of T-helper and a minority of T-suppressor/cytotoxic cells. All contrasted with the lymphomas, which showed Bcell monoclonality, markedly deranged T-subset proportions, or novel T-cell phenotypes. Although the main focus of this stud). was cases involving substantial populations of both B and T cells, preliminary observations were made in one case in which a pred o m i n a n c e o f T ceils a n d prominent epidermotropism simulated mycosis fungoides. Quantitative ultrastructural analysis in this case suggested a reactive T-cell process. Leu-6-positive Langerhans cells were increased in the epidermis and dermis in all five cases, and in the dermis they were found almost exclusively in T-cell compartments. It is proposed that this distribution is the anatomic correlate to the known functional role of Langerhans cells in antigen processing/presentation and T-cell activation. In the cutaneous " l y m p h node equivalent," Langerhans cells are analogous to interdigitating reticulum cells of reactive lymph nodes in distribution and, probably, in function. T h e DRC found in the germinal centers in two cases were probably antigenically identical and functionally afialogous to those in germinal centers of reactive lymph nodes, h n m u n o l o g i c phenotyping of serial cutaneous sections may aid in distinguishing reactive from neoplastic lymphoid lesions, lmmunoarchitectural analysis promises to be a powerful tool for the study of lymphoproliferative disease, ltust PATHOL 6:492--510, 1985.

The plethora of diagnostic tel-ms historically applied to ctHanet)us i)setldol)'lnl)homas (e.g., lymphocytoma cutis, lymphadenosis benigna cutis, Spiegler-

Received from tile Departnlcnts of l'athology, Internal Medicine (I)crmatology Section), and Biomedical Comnlunications, University of Arizona l leahh Sciences Center, Tucson, Arizona." Reviskm accepted for publication September 28, 1984. Address correspondtalcc and reprint requests to Dr. Wirt: Dep a r t m e n t of J:'athology, University o f Arizona t l e a h h Sciences Center, Tucson, AZ 85724.

492

MATERIALS AND METHODS

Clinical and routine histologic featttres for the five cases of ctttaneous i)seudolyml)homa are sutnmarized in table 1. For control studies clinically

IMMUNOARCHITECTURE OF CUTANEOUS PSEUDOLYMPHOMA (Wirt et all

normal skin fi'om a healthy subject (sun-exposed and unexposed, 0.3-cm punch biopsy specimens), reactive l y m p h nodes, T-cell lymphomas of skin and other organs, and B-cell lymphomas of skin and other organs were examined. Promptly after excision, sections of tissues were placed in OCT embedding medium (Miles Laboratories, Naperville, Illinois) and snap-frozen at - 15~0~ in isopentane quenclled in liquid nitrogen. The tissue blocks were then stored at - 7 0 ~ until sectioned. Multiple, serial ,t-lsm fi'ozen sections were cut fi'om ea'ch block, briefly dipped in acetone at room temperature, and stored at -20~ until imnmnohistochemical staining. For immunohistochenfical studies, B- and T-cell and DRC antigens w e r e detected in frozen tissue sections by a previously described immunol)eroxidase technique with monoclonal primary antibodies, 9 a biotin-conjugated secondary antibody, an a v i d i n horseradish peroxidase complex as the third stage, and diaminobenzidine tetrahydrochloride (I)AB) as the detection agent.10A 1 Briefly, frozen sections were fixed in 4~ acetone for 10 minutes and air-dried at room temperature. Monoclonal mouse antibody to Tcell subset antigens (Leu-1, -2a, -3a, -,t, -5, -6, -7; Uecton Dickinson, Mountain View, California); U-cell antigens L~ (BI and U2; Couher hnmunology, Hialeah, Florida); surface immunoglobulin (IgG, IgA, lgM, IgD, K, h; Bccton-Dickinson, Sunnyvale, California); DRC antigen (Dakopatts, I)enmark); HLADR (Ia) and CALLA (Becton-I)ickinson, Mountain View, California) was applied. Mouse ascitic fluid (Bethesda Research Laboratories, Gaithersburg, Maryhmd) was applied to one slide in each run instead of the primary antibody as a negative control. Following incubation and a phosphate-buffered saline wash (PUS, plI 7.4), a biotin-conjugated goat F(ab')_~-antimouse IgG antibody (Tago lnc, Burlingame, Califbrnia) was applied. Following incubation and a PBS wash, avidin-D conjugated with horseradish peroxidase (Vector l.aboratories, Burlingame, California) was applied. Following incubation and a I'BS wash, slides were immersed for 5 minutes in a DAB solution containing hydrogen peroxide (3 mg of DAB/ml PBS + 0. I ml of 30 per cent hydrogen peroxide). After PBS and distilled water washes, slides were immersed for 5 minutes in a copper sulfate solution (0.5 per cent copper sulfate in 0.85 per cent sodium chloride). After PBS and distilled water washes, slides were air-dried and then coverslipl)ed with Pro-Texx mounting metlium (I.erner Laboratories, New Haven, Connecticut). For cytochemical studies a-nal)llth)'ibutyrate esterase actMty, indicative of the presence of monocytic/histiocytic cells, was determined in frozen tissue sections as described previously. H Quantitative ultrastructural analysis of lynq)hocytic nuclear convolutions was p e r f o r m e d as described previously. 15 Briefly, the number of sharl)ly angulated nuclear invaginations in 100 l)'ntphocytes was scored in an electroTi microscol)e, and a histogram was preps{red. 493

RESULTS

Tile routine histologic findings in all five cases are summarized in table 1 . In cases 1 to 4 substantial populations of both B and T cells were present; compartmentalization of B and T cells, analogous to tlmt of reactive lymph nodes, was observed in these cases. The immunoarchitecture in cases 1 and 2 (figs. 1 to 5 and 7 to 10, respectively) was strikingly similar to that seen in reactive lymph nodes, one of which is illustrated in figure 6. In cases 1 and 2 distinct follicles with germinal centers, i.e., secondary follicles, were present, and ntmlerous DRC were observed in the germinal centers (fig. 4). The B cells in these follicles were polyclonal, with the expected K light chain predominance (figs. 5 and 10). Case 1 showed distinct mantle zone formation (figs. 1 to 3); in the follicle illustrated the nmntle zone lymphocytes can be divided into two zones: 1) an outer zone with phenotype B1 + B2- IgM + IgD + I g G - I g A - ; and 2) an inner zone with phenotype BI + B2 + IgM + IgD + IgG- l g A - . In case 2 a follicle with an Igl) + IgM + mantle zone (not illustrated) was also fi)und. Germinal centers showed predominantly intercellular and/or dendritic staining t)atterns for immtmoglobulin and B2, i.e., a lacy l)attern with an intercellular or dendritic appearance, in contrast to the distinct, peripheral circumferential rim of staining in nmntle zone cells, which probably represented surface antigen staining. However, some of the staining for immunoglobulin observed in germinal centers inay have been cell surface and cytoplasmic staining. 6-8 Since B2 antigen has been identified as the C3d receptor and C3d receptors are strongly expressed on DRC, the B2 staining of germinal centers probal)ly represented a combination of staining patterns, including DRC and B-cell staining (possibly intercellular staining of shed antigen for the latter). 16A7 For the imnmnoglobulin staining in germinal centers, the intercellular pattern ma)' have been staining of imnnmoglolmlin bound to DRC. 16 The phenotype of the germinal centers in cases 1 and 2 was B1 + B2 + IgM + IgG + (IgG- in case 2) IgD- IgA-. In case 1 the germinal center illustrated showed some intercelhdar and/or dendritic staining for IgD but was 1)redominantly negative in COml)arison with the adjacent mantle zone (fig. 1); the mantle zone showed some patches o f intercellular a n d ~ o r dendritic staining fox" IgG, but the staining was considerably less d'ense than that in the adjacent germinal center (fig. 3). In cases 1 and 2, Leu-6 + dendritic cells (Langerhans cells) effectively delineated the external border of follicles (figs. ,t, 9, and 10). In case 2 they were fotmd ~lmost exclusivel)' in tim interfollicular T-cell compartment, excluded ahnost entirel)' fi'om follicles. Case 1 showed marked expansion of secondary follicles, with Langerhans cells concentrated at the extreme peril)her )" of follicles. The T-cell zone in case 1 was not as large or distinctly delineated as that in case 2 but was identilied as a band of cells adjacent to the peripheral rim of l.angcrhans cells (fig. 4); in another section flom case I (not illustrated)

HUMAN PATHOLOGY TABLE 1.

Volume 16, No. 5 (May 1985]

Clinical a n d Histologic Features

Lesion:

Site~Appearance~ Case

Patient's Age (yr)/Sex 24/F

42/F

I)urationtBiol)sy Data

Other l'ertinent If;story

Recurrent red papules and plaques, face, neck, forearms (since childhood); shave biopsy fiom nose, 0.2 cm deep 1.5-cm red plaque, left chin (19 too); elliptical biopsy, 3.0 x 1..t x 0..t cm

Photosensitive; malar involvement; ANA. 9t0; hontogeneous patterll

Routine I listologic Findings

CollullelltS

i'olymorphous lymI~hohistiocytic infiltrate with germinal center formation; g r e n z Zolle 1)resent,

no epidermotropism Polymorphous l)'mphohistiocytic infiltrate with germinal center formation; grenz

ZOIIC

Clinically recurrent in sanle area a few months after excision

present,

no epidermotrolfism

(fig. 7) 3

54/F

Recurrent red lmpules and nodules on trunk and arms, up to 2 clll (6-7 yr); punch biopsy, upper back, 0.3 cm in diameter, 0.4 cm deep

tlistory of l)mplmma, left inguilml lymph node biopsy in 1965 interpreted (in 1978) as diffuse histiocytic lyml~homa; no therapy from 1965 to 1976; left inguimd l)'mph node biopsy in 1976 interpreted as tliffitse mixed lymphoma; staging negative except fi~r skin lesions interpreted as secondary l)mphoma; CIIOP and B('G therap)" in 1976 with CR; recurrent skill lesions: in 1977 with additional dlelllOtllerapy and CR; in 1980 and 1981 with radiation therapy and CR; in 1982-1983 with intralesional steroid injection and CR; otherwise in good healtll with no

a m o r e sharply d e f i n e d T-cell zone was present external to a tollicle. As in the reactive lymph node illustrated in figure 6, the T cells were far less num e r o u s in the mantle zone than in tile adjacent Tcell zone or germinal center. T h u s , case 1 showed a vestigial T-cell zone relative to the d e v e l o p m e n t o f secondary follicles, while cas'e 2 showed tile opl)osite pattern, with a large interfollicnlar T-cell zone containing m o d e r a t e l y sized follicles (fig. 8). In both cases .1 and 2, T cells a d m i x e d in the gernfinal center were o v e r w h e h n i n g l y o f the Leu-3 + h e l p e r l ) h e n o t y p e , with rare Leu-2 + supprcssor/cytotoxic cells. T h e interfollicular portion o f the infiltrate in case 2 consisted p r e d o m i n a n t l y o f T cells, which stained strongly fi)l- tile pan-T-cell antigens Leu-I and Len,t (figs. 8 and 10). T h e majority stained weakly for Leu-5 (E-rosette r e c e p t o r antigen). In the interfollicular T-cell zone f r o m case 2 and in the vestigial T -

1982 skin biopsy: polyluorplIous I)'lnpllolfistiocytic infihrate, dense and diffuse in upper dermis, nodular in lower dermis; no germinal centers apparent; grenz zone present, minimal epitlernlotropism (fig. I1)

1965 lymph node biops)" unavailable for review; review of 1976 lymph node biopsy: non-llodgkin's I)mpholna, favor nodular mixed; review of 1976 skin biopsy: polymorphous lyml,hohistiocytic infiltrate silnilar in distrilmtion to 1982 biOl)S)', but with nodular aggregates of large lymphoid cells surrounded by small lymphocytes, suggestive of tollicular architecture with germinal Cellters

cell zone f r o m case 1, l.cu-3 -~ h e l p e r cells predonfinated over Leu-2 + SUl)pressor/cytotoxic cells. T h e innnuno,lrchitectural features o f tile reactive iynq)h nodes ilhtstrated in figure 6 were ver)' sinlilar to those observed in cases 1 and 2. B cells external to the nmntle zone were we,lkly B2 *, while the mantle zone lyml)hocytes were B2 +. Mantle zone cells h a d the p h e n o t y l ) e B1 + B2 + K + L + IgD + IgM + I g G - I g A - . T h e germilml c e n t e r was strikingly negative for IgD and showed an intercelhllar and/o,,l~ dendritic staining pattern for B1, B2, IgG, and IgM. In addition, scattered lg(, ~ I)lasmacytoid a n d p l a s m a cells w e r e i d e n t i f i e d in tile g e r m i n a l center, mantle zone, and interf'ollicnlar T-cell zone. Germinal centers contained iltllllerotls DRC and a d m i x e d T cells, overwhehningl)' o [ the Leu-3 + helper p h e n o t y p e (not ilhlstrated). T h e interfollicular zone consisted predonfinantly o f T cells, ahhottgh signifi-

494

IMMUNOARCHITECTUREOF CUTANEOUSPSEUDOLYMPHOMA(Wirt et al.) TABI.E t. Continued

Lesion: Site/Appearancel

Case

Patient's Age (yr)/Sex

Duration/Biopsy Data

Other Pertinent History

Routine ttistologic Findings

Comments

constitutional symptoms, lymphadenopathy, or hepatosplenomegaly; abdonfinal CT scan and bone marrow in 1982 ilornlal

5

69/M

5 x 4-cm red plaque, right forehead (10 yr); ptmch biopsy, 0.4 cm in diameter, 0.4 cm deep

Lymphohistiocytic infiltrate with scattered plasma cells, diffilse, no germinal centers apparent; grenz zone present, no epidermotropism

67/F

2-cm red paptfle, right posterior shoulder (5 too); elliptical biopsy, 4.5 x 2.5 • 0.7 cm

Lymphohistiocytic infiltrate with scattered plasma cells, rare esoinophils, and lichenoid distribution; prominent epidernmtropism; minimal spongiosis (fig. 16)

Two additional smaller plaques subsequently developed (one each on forehead and nose); large plaque on forehead rebiopsied; routine histologic features: subpopulation of large pleomorplfic lyntphoid cells, in addition to small lymphoc)tes seen previously; hint of compartmental architecture on frozen sections stained with hematoxylin-eosin; staging negative (abdominal CT scan, chest radiograph, bone marrow biops)'); CHOP and PVB therapy with CR Small lymphoid cells without substantial pleomorphism in epidermis and dermis, often associated with histiocytoid cells suggestive of Langerhans cells (Figure 16).

ABBREVIATIONS:CR, complete respouse; CtlOP, cyclophosphanfide, Adriamycin, Oncovin, prednisone; BCG, bacille Calmette-Gufrin vaccine; PVB, cisplatin, vinblastine, bleomycin.

cant n u m b e r s o f I g D + IgM + BI + weakly B2 + cells were also present. In the interfollicular T-cell zone o f this particular reactive l y m p h node, the Leu-3/Leu2 ratio was very high (estimated p r o p o r t i o n o f Leu3 + cells, g r e a t e r than 90 p e r cent). T h e p o l y m o r p h o u s l y m p h o i d infiltrate in case 3 was " t o p he~wy," i.e., d e n s e and diffuse in the upl)er dermis a n d patchy a n d n o d u l a r in the lower dermis, with no g e r m i n a l c e n t e r f o r m a t i o n a p p a r e n t on routine histologic e x a m i n a t i o n (fig. 11). H o w e v e r , imm u n o a r c h i t e c t u r a l analysis s h o w e d c o m p a r t m e n t a l architecture o f B a n d T cells (figs. 12 a n d 13), alt h o u g h the c o m p a r t m e n t s w e r e less sharply d e f i n e d than in cases 1 and 2. T h e B-cell p o p u l a t i o n stained the s a m e for B 1 a n d B2, i.e., a b o u t the s a m e n u m b e r of cells stained in the s a m e areas with the s a m e (peripheral) pattern, h n m u n o g l o b u l i n was detected on a m i n o r i t y o f t h e s e cells, q ' h e i m m u n o g l o b u l i n -

b e a r i n g cells w e r e p o l y c l o n a l , with tile e x p e c t e d K p r e d o m i n a n c e (fig. 12). A b o u t the s a m e n u m b e r s o f cells stained for I g D a n d for light chains. Rare IgMpositive cells were present, but no I g G staining was seen. T h u s , a m i n o r i t y o f B cells had the p h e n o t y p e BI + B2 + I g D +, a n d a m a j o r i t y o f B cells h a d the p h e n ~ t y p e B1 + B2 + S I g - . A minority o f cells were also weakly C A L L A + . T i m i m m u n o a r c h i t e c t u r e in case 4 was very similar to that in case 3. A hint o f follicular a r c h i t e c t u r e was o b s e r v e d , on r o u t i n e histologic e x a m i n a t i o n in case 4, but i m m u n o a r c h i t e c t u r a l analysis showed distinct c o m p a r t m e n t a l i z a t i o n o f B and T cells (fig. 1,t). T h e large blastic cells identified on routine histologic ex.anfination were B cells, the majority o f the i m m u n o t y p e B I + B 2 - S I g - . A minority o f cells e x p r e s s e d immunoglobulin. These imnmnoglobulin-bearing cells w e r e p o l y c l o n a l , with K light c h a i n p r e d o m i -

495

HUMAN PATHOLOGY

Volume ~6, No. 5 (May '1985]

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FIGURE 4 [top]. Case '1. Serial sections of skin stained for ~-naphthylbulyrate esterase (ANBE] [left) and IgD [right]. The immunoarchitect i r e of the dermal infiltrate is strikingly similar to that of a secondary follicle in a reactive lymph node, with a germinal center asymmetrica!ly located to the left and a mantle zone to the right. The small lymphocyte population of the mantle zone is strongly IgD+. The germinal center shows some intercellular and/or dendritic staining for IgD., but is predominantly IgD-0 unlike the mantle zone. Numerous ANBE ~ histiocytes are scattered in the large-cell population of the germinal center. [ x 80.] FIGURE 2 (bottom]. Case 1. Serial sections of skin stained for B1 [left) and B2 [right]. The entire follicle is BI +, while B2 staining is more restricted9 The germinal center shows strong intercellular and/or dendritic B2 staining9 A contiguous rim of small mantle zone lymphocytes is also B2 +, and the mantle zone can be divided into two zones: an outer zone that is IgD § B1§ B2-, and an inner zone that is IgD § B1§ B2 ~" [the arrowhead marks the border between these two zones]. This immunoarchitecture strongly suggests zonal maturation of lymphoid cells and supports the same pattern of B-cell trafficking and maturation proposed for the reactive lymph node illustrated in figure 6. I x 80.]

496

IMMUNOARCHITECTURE OF CUTANEOUS PSEUDOLYMPHOMA (Wirl et ol.)

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FIGURE 3 (top). Cose 1. Seriol sections of skin stoined for IgM [left) end IgG [right). The montle zone cells ore IgM +, with o peripherol [cell suffoce) stoining paffern. The germinol center shows predominon'fly intercellulor end/or dendritic stoining for IgM e n d IgG. The montle zone shows some intercellulor end/or dendritic stoining for IgG, but the staining is considerobly less dense then thol of the o d j o c e n t germinol center. ( x 80.) FIGURE 4 (bottoml. Cose 1. Serial sections of skin stained for Leu-4 [left), Leu-6 [middle), end dendritic reticulum cells [Den) (right). Numerous dendritic reticulum cells ore present in the germinol center, while numerous Leu-6* Longerhons cells rim the follicle e n d ore e!most entirely excluded from if (see also figs. 9 (and 10). The doric oblique line in theTecfion sfoined for Den represents c~n immufoble scratch In the gloss underlying on irreploceoble section. This distribution of dendritic reticulum cells e n d Longerhons cells in cutoneous p s e u d o l y m p h o m o Is highly suggestive of the distribution of reticulum cells in reoctive lymph nodes, w h e r e T- end B-cell comportments each have distinct populations of reticulum cells. The T-cell zone is identified as c) b a n d of cells edjc]cent to the peripherol rim of Longerhons cells. The numerous Leu-4 + T cells a d m i x e d in the germinol center ore Iorgely Leu-3 + helper cells, with rore Leu-2 § suppressor/cyfotoxic cells9 The presence of Leu-3 § helper cells In the B-cell zones of cutaneous pseudolyrnphomc] probably represents the hlsfologlc correlofe of B-cell d e p e n d e n c e on T cells for response to certoin onfigens. (Left, x 54; middle, x 54; right, x (50.)

497

HUMAN PATHOLOGY

Volume 16, No. 5 [May 1985)

I,

FIGURE 5. Case 1. Serial sections of skin (follicle different than that illustrated in figs. 1 to 4) stained for z population is polyclonal, with the expected K predominance. ( x 137.l

nance; the heavy chains expressed were IgD and IgM; no staining for IgG, IgA, or CALLA was observed. T cells admixed in the B-cell compartments in both cases 3 and 4 were predominantly of the Leu3 + helper phenotype, with rare Leu-2 + suppressor/ cytotoxic cells. Leu-3 + cells predominated over Leu2 + cells in the T-cell compartments in both cases. In both cases the densities of Leu-6 + Langerlmns cells in the epidermis and dermis were m o d e r a t e l y to markedly increased, and in the dermis these cells were found chiefly in T-cell compartments (figs. 13 and 15). No D R ( were detected either in case 3 or 4, or in normal skin biopsies. The lichenoid, epidermotropic infiltrate in case 5 (fig. 16) was composed predominantly of T cells, with densely admixed Leu-6 + Langerhans cells and a small minority o f polyclonal B cells (BI + B 2 IgD+, ' weakly IgM +, with scattered IgG + plasma cells). No compartmentalization of these B cells was detected. T h e lichenoid infiltrate f r e q u e n t l y obs'cured the d e r m a l - e p i d e r m a l j u n c t i o n . Sections stained with hematoxylin-eosin showed solitary and small groups of small lymphoid cells in the epidermis, frequently associated with histiocytoid cells with pale staining elongated nuclei suggestive of L'angerhans cells (confirmed as Langerhans cells by immunohistochemical staining for Leu-6 antigen). Leu3 + cells predominated moderately over Leu-2 + ceils 498

(left)

and

h (right).

The B-cell

in the dermis and epidermis, in approximately the same ratio in tile two locations (fig. 16). Ahhough Leu-3 + cells predominated over Leu2 + cells in the T-cell conapartments in all cases, there was moderate variability in tile ratios. In tile reactive lymph node chosen fi)r illustration in this study, tile ratio was very high (estimated proportion of Leu-3 + cells, greater than 90 per cent; clinical follow-up data gave no reason to d o u b t this as a reactive lymph node). It is difficult to quantitate T-cell subset ratios accurately in tissue section, and more precise quantitation is deferred to cell suspension (flow cytometric) studies. The T-cell compartments in all cases shared certain o t h e r characteristics. T h e majority of cells stained strongly with the pan-T-cell antibodies Leu-1 and Leu-4, and the majority of cells stained weakly to moderately with Leu-5, i.e., antibody to E-rosette receptor antigen. Rare Leu-7 + (natural killer) cells were scattered in the infiltrates. Although it was our distinct impression that many of the T cells were Ia + (i.e., acnvated), double immunostaining experiments would be necessary to prove this impression. Quantitative ultrastructural analysis in cases 1, 3, 4, and 5 revealed that a relatively large percentage of the lymphocytes (30 to 59 per cent) had no distinct nuclear invaginations, similar to the range seen in control subjects (19 to 55 per cent), but unlike find-

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FIGURE 6. Serial sections o f a r e p r e s e n t a t i v e r e a c t i v e l y m p h n o d e s t a i n e d for Leu-4 ( t o p left), B2 ( m i d d l e t o p ) , BI ( t o p r i g h t ] ; I g G ( b o t t o m bottom], and IgM (bottom r i g h t ] . The c a p s u l a r s u r f a c e of t h e l y m p h n o d e is p r e s e n t a t t h e t o p o f e a c h f r a m e . The left], I g D [ m i d d l e c o m p a r t m e n t a l i z a t i o n o f l y m p h o c y t e s u b p o p u l a t i o n s is r e a d i l y a p p a r e n t . The interfoTlicular a r e a s consist p r e d o m i n a n t l y o f T cells, a l t h o u g h significant n u m b e r s o f IgD + I g M + B1 § w e a k l y B2 + cells a r e a l s o present. The p h e n o t y p e o f m a n t l e z o n e ceils is IgD + I g M + I g G - B1 + B2 +. The g e r m i n a l c e n t e r is a l m o s t t o t a l l y I g D - a n d shows a p r e d o m i n a n t l y intercellular a n d / o r d e n d r i t i c s t a i n i n g p a t t e r n for BI, B2, IgG, a n d IgM. N u m e r o u s d e n d r i t i c r e t i c u l u m cells a r e p r e s e n t in g e r m i n a l c e n t e r s (not illustrated]. I g G + p l a s m a cells a r e s c a t t e r e d in t h e g e r m i n a l center, m a n t l e zone, a n d interfollicular areas. The interfollicular T cells a n d t h e n u m e r o u s T cells in t h e g e r m i n a l c e n t e r a r e p r e d o m i n a n t l y of t h e L e u - 3 + h e l p e r p h e n o t y p e , w i t h a small m i n o r i t y o f L e u - 2 § s u p p r e s s o r / c y t o t o x i c cells. In this p a r t i c u l a r r e a c t i v e l y m p h n o d e t h e Leu3/Leu-2 ratio in t h e inteflollicular z o n e is p a r t i c u l a r l y high, [ e s t i m a t e d p r o p o r t i o n of Leu-3 + cells, g r e a t e r t h a n 90 p e r c e n l } . The i m m u n o a r c h i t e c t u r e Is v e r y similar t o t h a t o b s e r v e d in c a s e s ~ a n d 2 o f c u t a n e o u s p s e u d o l y m p h o m a a n d suggests t h a t u n a c t i v a t e d B cells o u t s i d e follicles (IgD + I g M + I g G - BI + w e a k l y B2 +) m a t u r e t o full B 2 - p o s i t i v i t y i n t h e m a n t l e zone, w i t h s u b s e q u e n t i m m u n o b l a s t i c transf o r m a t i o n a n d h e a v y c h a i n s w i t c h i n g in t h e g e r m i n a l c e n t e r . [ T a p , x b l ; b o t t o m , x69.]

l ) s e u d o l y m p h o m a g r o u p with m o r e than eight nuclear invaginations.

ings in, patients with mycosis f u n g o i d e s (fig. 17). In the mycosis f u n g o i d e s g r o u p the p e r c e n t a g e o f lymphocytes with no distinct nuclear invaginations was relatively small (3 to 15 p e r cent). For o n e to seven n u c l e a r i n v a g i n a t i o n s , t h e r e was m o r e o v e r l a p between the control a n d p s e u d o l ) ' m p h o n m g r o u p s than between the pseudolyml)homa and mycosis fungoides g r o u p s . Unlike the control a n d mycosis timg o i d e s g r o u p s , t h e r e w e r e n o l y m l ) h o c y t e s in t h e

D I S C U S S I O N

We have achieved substantial u n d e r s t a n d i n g o f tile c o m p l e x i t m n u n o l o g ) ' o f S A L T a n d c u t a n e o u s p s e u d o l ) ' n q ) h o m a by the m e t h o d o f serial tissue sec-

4 9 9

HUMAN PA1HOLOGY

Volume 16, No. 5 [May 1985]

FIGURE 7. Case 2. Polymorphous lymphohistiocytic dermal infiltrate with germinal center formation (arrow] evident on sections stained

with hematox~in-eosin. [Left, x 60; right, x 250.]

tion immunoarclfitectural analysis with monoclonal antibodies to T- and B-cell and DRC antigens. This method allows phenotypic analysis while maintaining arclfitectural relationships. Ahhough the analysis is static, this method can be used to make inferences about the dynamic processes of cell trafficking and zonal maturation. Cutaneous pseudolymphoma emerges as a reactive, hyperplastic proliferation of SALT, with immunoarclfitectural features frequently analogous to those of reactive lymph nodes and with subtle immunologic evidence of aberrance identified in some cases. Even when the infihrate appears diffuse by routine microscopy, immunoarchitectural analysis may reveal an underlying compartmental architecture of B and T cells. O u r cases of cutaneous p s e u d o l y m p h o m a invo.lving substantial populations of B and T cells, the main focus o f this study, d e m o n s t r a t e d compartntental architecture suggesting clonal expansion of B and T cells in separate domains. T h e immunoarcifitectural features in these cases were analogous, sometimes strikingly so, to those found in reacti;.'e lymph nodes 6-8 (unpublished observations). In two cases the B-cell c o m p a r t m e n t s showed germinal center formation, arid in one case distinct mantle zone formation was observed. As in reactive lymph

nodes, the B-cell conlpartments were polyclonal, with the expected predominance of K- over X-bearing cells, and tile T cells admixed in tile B-cell compartnlents were overwhelmingly of tile helper phenotype. In the T-cell compartments in all of our cases of cutaneous p s e u d o l y m p h o m a , helper cells predominated over suppressor/cytotoxic cells, and, although the ratios showed moderate variability, the markedly deranged proportions or novel phenotypes evident in cutaneous T-cell lymphoma 18--~ were not found. Substantial variability in helper to suppressor/cytotoxic ratios was reported previously for benign and malignant cutaneous lymphoid infihrates, I'~suggesting that tiffs criterion alone cannot be used to distinguish berrign from malignant lesions because of possible overlap, especially in cases in wlfich T cells predominate. However, the demonstration of an immunoarchitecture in c u t a n e o u s p s e u d o l y m p h o m a that is analogous to that o f reactive iyn~phadenopathy speaks t01" a reactive process, reducing concern about helper to suppressor/cytotoxic ratios in these cases. In fact, the clearly reactive l}'mpll node chosen for illustration in this study had a very high helper to s_uppressor/cytotoxic ratio in the interfollicular T-cell zone.

O u r cases o f cutaneous pseudolyntphoma and reactive lymphadenopatl W support the concept that 500

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a l m o s t e x c l u s i v e l i' i n T - c e l l c o m l m r t n l e n t s , b e i n g almost entirely excluded from B-cell compartments. ._This distribution is a n a l o g o u s to that of interdigirating reticuhun cells in lymph nodes, and we suggest that Langerhans cells in SALT are the functional equivalent of interdigitating retictdum cells in lymph

the T- and B-cell compartments of SALT and lyn!ph nodes each have immunologicall)' distinct types of ret i c u l u m cells.'22. 23 S p e c i f i c a l l y , o u r c a s e s d e m o n s t r a t e " that the distribution of Langerhans cells in the dermis has a consistent padern in relation to the compartments of li'ml)hoid cells. Langerhans cells are found 502

IMMUNOARCHITECTUREOF CUTANEOUSPSEUDOLYMPHOMA(Wirtet al.)

FIGURE 11. Case 3. Dense, polymorphous infiltrate ('1982 skin biopsy) that appears diffuse in sections stained with hematoxylin-eosin, i.e. compartmental architecture is not apparent. (Top, x96; bottom, x4'12.]

nodes. T h e flmctional equivalence o f Langerhans ceils and interdigitating reticulum cells was proposed previously in a study in which both cell types were found in l)'mph nodes of patients with dernmtopathic lymphadenopathy. -~~ However, the anatomic compartmentalization of Langerhans and other cells that we have d e m o n s t r a t e d iLt c u t a n e o u s pseudolymphoma is strong evidence that the skin can bc the site of a complete lymphoid organ, rather than merely

part of the af,f,erent limb for a regional I)'mph node, in which tile immune response is centralized. The proposal that skin is potentially a complete lymphoid 9organ is consistent with previous observations and speculations about SALT. 25-27 Langerhans cells are known to be the epidermal cells responsible for processing and presenting antigen to T cells, as well as for activating T cells. In a recent review of Langerhans cell structure and function, Wolff and Sting128 503

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FIGURE 12. Case 3. Serial sections o f skin stained for B2 (top left), keu-1 (bottom left), ~ (top right), a n d X (bottom right). Although the d e r m a l Infiltrate a p p e a r s diffuse o n sections stained with hematoxylin-eosin, definite T- a n d B-cell c o m p a r t m e n t s a r e present, a l t h o u g h less sharply d e f i n e d than those in the other cases. As in reactive lymph nodes a n d in cases 1 a n d 2 of cutaneous p s e u d o l y m p h o m a , T cells a r e a d m i x e d in the B-cell zones, overwhelmingly o f the Leu-3 + helper p h e n o t y p e ; Leu-3 § h e l p e r cells also p r e d o m i n a t e over Leu2 + suppressor/cytotoxic cells in the T-cell zones. The minority of B cells that express immunog!obulin a r e polyclonal, with the e x p e c t e d p r e d o m i n a n c e . A majority of B cells d o not express immunoglobulin, a n d no dendritic reticulum cells are f o u n d in B-cell zones. Thus, a l t h o u g h there is s o m e subtle i m m u n o l o g ! c e v i d e n c e o f a b e r r a n c e , t h e r e is n o e v i d e n c e of m o n o c l o n a l proliferation, a n d the previous diagnosis o f c u t a n e o u s l y m p h o m a [ m a d e on sections from this biopsy stained with hematoxylin-eosin] c a n n o t b e substantiated. [Left, x 80; right, x 274.)

cells and DRC nlay have a function in organizing the T - and B-cell conipartnlents, respectively. Skin a n d lnucosal surfaces are u n i q u e in that khey ;ire directly exposed to environnlental antigens. A h h o u g h the responsible antigen frequently cannot be i d e n t i f i e d in c u t a n e o u s p s e u d o l y n l p h o m a , the identity or association is known in some cases (e.g., insect, viral, p l a n t , t a t t o o - d y e , a n d d r u g antigens). '-'9''3'~ It inay be that the " d e r m a r ' lynlph node equivalent forms in the context o f antigenic persistence a n d c h r o n i c a n t i g e n i c stimulation, with Langerhans cells nligrating to tile dermis, where they are normally very sparse. With f u r t h e r antigenic persistence and stiinulation, L a n g e r h a n s cells ilia}' migrate with e n t l - a p p e d a n t i g e n to regional l y m p h nodes, causing (lernlatol)athic lynli)hadenopathy. T h e Inor-

stated that: "At present, it is still u n k n o w n where LC (Langerhans c e l l ) - - T - l y n l l ) h o c y t e interactions occur in viva and whether, indeed, such interactions are physically required t a r T-cell s timulatory signals9 LCs coukl travel to the lyml)h nodes, where L C - T - l y i n phocyte e n c o u n t e r s could take place, but it is also possible that the snlall immbers o f lynil)hocytes usually present in skin suffice for the generation (If stimulatory signals." O u r findings strongly suggest that this interaction between L a n g e r h a n s cells and T cells can occur in the skin. T h e n u n l e r o u s DRC f o u n d in the g e r i n i n a l centers in two o f o u r cases o f cutaneous pseudol)'nlphoma were probably antigenically identical and timctionall)' analogous to the DRC in the germinal c e n t e r s o f . r e a c t i v e lyniph nodes. T h e Langei-hans 504

IMMUNOARCHIIECTURE OF CUTANEOUS PSEUDOLYMPHOMA (Wirt et al.]

pholog-ic variations in cutaneous p s e u d o l y m p h o m a may reflect the diversity of stimulating antigens, with differences in i m m u n e response to different antigens, variation in response of different hosts to similar antigcns, or examination of an evolving process at different times. With one antigen, both B- and Tcell compartments may form, while another might stimulate T-cell clonal expansion alone. T h e zonal pattern o f immunoglobulin, B 1, and B2 expression in case 1 warrants fl~rther comment. The static images in this case and in our cases of reactive lymphadenopathy suggest a dynamic process of B-cell activation, clonal expansion, and differentiation. Our speculations regarding zonal maturation of B cells in cutaneous pseudolynaphoma and in reactive lymph nodes are in keeping with the temporal sequence of B-cell surface marker expression described in flow cytometric studies after activation in vitro. 36 These studies show that B2 is expressed only weakly on peripheral blood B cells but that these cells transiently become B2 + after activation. The loss of B2 that occurs four to five days after activation is accompanied by loss of surface IgD. The loss of B I that occurs six to seveu days after activation is correlated with the acquisition o f surface and cytoplasmic IgG. T h e immunoarchitecture of the reactive lymph node illustrated in figure 0 suggests that unactivated B cells external to follicles (lgD + IgM + BI + weakly B2 +) mature to full B2-positivity in the mantle zone, with subsequent immunoblastic transformation and heavy chain switching in the germinal center (evidenced by loss of IgD and acquisition of cytoplasmic immunoglobulin, preclominantly IgG). The phenotype of the B cells external to follicles is that of peripheral blood B cells. T h e staining pattern of the germinal center for B2 may reflect a combination of events: 1) loss of B2 antigen from cell surfaces but persistence in the intercellular space (i.e., intercellular staining); and/or 2) staining o f I)RC. Recent studies indicate that the B2 antigen is the C3d receptor on B cells and that C3d receptors are strongly expressed on DRC. 16,]7 Either or both circumstances would explain the B2-positivity of germinal centers when the cell suspension studies predict that the IgD- germinal center B cells should also be B2-.36 T h e immunoarchitecture in case 1 of cutaneous pseudolymphonm is strikingly similar to tlmt of a seconclary follicle iu a reactive lymph node and supports the same pattern of B-cell trafficking and maturation proposed earlier9 In this case the mantle zone illustrated can be divided into two zones, an outer zone that is IgD + IgM + BI + B2- and an inner zone that is lgD + IgM + BI + B2 +. The outer mantle zone lymphocytes are probably at the maturational stage of unstimulatecl peripheral blood B ceils, and the outer area of the mantle zone is probably a staging area for B cells not yet stimulated by antigen. IgI)-positivity iclentifies a B cell prior to antigenic stimulation. ~6 With exposure to antigen, B2 expression increases (inner mantle zone cells, B2+). Subsequcntly, IgD is 505

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FIGURE 13. Case 3. Serial sections o f skin stained for B2 ( f o p ] , Leu-6 [ m i d d l e l , a n d Leu-I [ b o t t o m ) . Numerous Leu-6 § Langerhans cells a r e present'Tn the epidermis a n d dermis. In t h e dermis the Langerhans cells a r e f o u n d p r e d o m i n a n t l y in the T-cell zones a n d c o n c e n t r a t e d a t the borders of T- a n d B-cell zones, but are exc l u d e d from the latter a l m o s l entirely [an asterisk marks the s a m e "dilated vessel in all three sections; in addition, in the section stained for r eu-6, asterisks to either side of the vessel mark t w o B-cell zones]9

[x58.]

HUMAN PATHOLOGY

Volume 16, No. 5 (May 1985]

Case 4. Serial sections of skin stained for Leu-1 [left] and B1 [right]. Distinct compartmentalization of T a n d B cells b e c a m e FIGURE 14. a p p a r e n t on serial tissue section immunohistochemistry. This case is similar to case 3 in that a minority of B cells express immunoglobulin, and these are polyclonal; the majority of B cells d o not express immunoglobulin, a n d dendritic reticulum cells are not found in B-cell zones. The subtle immunologic evidence of aberrance found in cases with no evidence of monoclonality m a y prove useful in identifying a group at risk for malignant transformation. ( x 63.)

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IMMUNOARCHITECTURE OF CUTANEOUS PSEUDOLYMPHOMA [Wirt et al.)

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Case 5. Sections of skin stained with hematoxylin-eosin (fop,/eft and right), and for Leu-4 (bottom left) and Leu-2 (bottom right). The epidermotroplc, nonspongiotic, predominantly T-cell infiltrate in this case is of the histologic type that must be distinguished from early mycosis fungoides. The central rete ridge [top left) is also illustrated at higher magnification [top right): numerous small FIGURE i6.

lymphocytes, frequently associated with histiocytoid cells suggestive of Langerhans cells [confirmed on the Leu-6-stained section], have infiltrated the epidermis. The infiltrate is c o m p o s e d predominantly o~ T cells. The T cells infiltrating the epidermis are c o m p o s e d of both Leu-2 § suppressor/cytotoxic cells [bottom right) and Leu-3 § helper ceils [not illustrated, but a p p r o x i m a t e d by mentally subtracting Leu-2 from Leu-1]. The Leu-3/Leu-2 ratios are approximately lhe same in the dermis.and epidermis 9 The clinical, immunologic, a n d quantitative ultrastructural evidence in this case supports a reactive, rather than a malignant, process. [Top left, • 240; top right, x 845; bottom left, x 240; bottom right, x 240.1

507

HUMAN PATHOLOGY

Volume 16, No. 5 [May 1985)

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FIGURE t7. Percentages of lymphocytes with various degrees of nuclear convolution in dermal infiltrates from control subjects and from patients with pseudolymphoma and mycosis fungoides. Quantitative ultrasfructural analysis of cases i, 3, 4, and 5 supports the diagnosis of reactive, rather than malignant, lymphoid Infiltrates. In these cases a relatively large percentage of lymphocytes have no distinct nuclear invaginations, similar to the range seen in control subjects, and in contrast to findings in patients with mycosis fungoides. In the mycosis fungoides group, the percentage of lymphocytes with no distinct nuclear invaginations is relatively small. White bars, control group~ black bars, pseudolymphoma group; crosshatched bars, mycosis fungoides group.

cases. In case 3 the B-cell population stained the same for B 1 and B2, and immunoglobulin was detected on a minority of these cells. Thus, a minority of B cells had the plienotype B1 + B2 + IgD + (with polyclonal light chain expression), and a majority of B cells had the p h e n o t y p e B I + B2 + S I g - . T h e exact maturational stage of the latter cells is uncertain, but we suspect that they may represent immunoblasts (transformed, activated B cells) tlhat have lost surface immunoglobulin but retained B2. The very long clinical course of recurrent skin lesions without any evidence of systemic disease supports the diagnosis of a nonneoplastic condition in this case. Case 4 also illustrates several points of importance. Initial biopsy of a large plaque that had been present on this patient's forehead for ten years revealed a nonelfidermotropic, predominantly T-cell infihrate, extending deep into the dermis. In tiffs initial biopsy specimen, imnmnoarchitectural analysis revealed a single B-cell follicle at the deep margin of the specimen. After two additional, smaller facial t~laques d e v e l o p e d , re-biopsy o f the large plaque showed a population of large, pleomorphic, blasticappearing cells, with only a lfint of compartmental architecture on routine histologic examination. However, i n ~ m n o a r c h i t e c t u r a l analysis o f this second specimen showed distinct compartmentalization of B and T ceils. The large, blastic-appearing cells were B cells with an immunotype consistent with immunoblasts (B 1+ SIg-). A minority of cells expressed surface immunoglobulin; these cells may have been B cells prior to immunoblastic transformation. Alt h o u g h a l)athologic diagnosis o f malignant lym-

lost, indicating heavy chain switching. As in reactive lymph nodes, the germinal center in case 1 shows intercellular and/or dendritic B2 staining. Collectively, our findings suggest zonal B-cell maturation in this case of cutaneous pseudolymphoma, with B2 acqtfisition a n d suspected loss, and IgD expression and loss. As in reactive lymph nodes, the presence of Leu3 + helper cells in the germinal centers of cutaneous pseudolymphoma probably represents the histologic correlate of B-cell dependence on T cells for the response to certain antigens. These helper cells appear to be ideally located to drive B-cell differentiation at the point of heavy chain switclfing. Cases 3 and 4 require filrther comment because they illustrate the diagnostic difficulties that can be encountered in extensive cutaneous lymphoid infiltrations. In both cases some subtle immunologic evidence of aberrance was found, but the weight of evidence in both points to a reactive/aberrant rather than a malignant process. The rectu'rent skin lesions in case.3 were repeatedly di~'lgnosed as cutaneotls lymphoma by routine Ifistologic examinations during a period of several years, but the immunoarcllitecture o f one lesion supports the diagnosis of pseudolymp h o m a rather than malignant lymphoma (figs. 12 arid 13). The dermal infihrate was composed of approximately equal proportions of B and T cells, with numerous Langerhans cells. Ahhotlgh the infihrate appeared diffnse on rontine histologic examination, the B-, T-, and Langerhans cell populations showed d e f n i t e compartmentalization, ahhougll these compartments were not as sharply defined as in the other 508

IMMUNOARCHITECTURE OF CUTANEOUS PSEUDOLYMPHOMA (Wirt et al.)

pltoma could not be m a d e in tiffs case, tire patient was treated with combination c h e m o t h e r a p y on tire basis of clinical findings. T h e response of the lesions to this therapy does not speak for or against either a benign or malignant process. T h e sccond biopsy was significantly d e e p e r than tire first and revealed a B-cell process only suggested in the first biopsy, e m p h a sizing the importance of deep biopsy in tltese cases. In c,'3tes 3 and 4 no DRC w e r e found in the infiltrates, suggesting the possibility of some degree o f B-cell autonomy. Thus, although there was no evidence of monoclonal proliferations in these two cases, there was sonte subtle immunologic evidence of aberrance, perhaps related to antigenic persistence and/or abnormal host i m m u n e response. In cases 3 and 4 we cannot rule out the possibility tlmt a k light chain-restricted monoclonal population was not detected because of l)olymorl)hism tlmt was not detected by the monoclonal anti-h reagent used. 37 late also cannot rule out the possibility that the infiltrates in these cases r e p r e s e n t e d in~munoglobttlinnegative B-cell lympllomas. Althougll immunoglobu l i n - n e g a t i v e B-cell l)'tnplaomas have been well described, 3s the inability to d e m o n s t r a t e i m m u n o globulin by conventional means in a B-cell l)opulation does not specifically indicate malignancy. We suspect that a sintilar situation may occur in reactive/aberrant proliferations. Perhaps thee only way to resolve the issue o f clonality in these two cases would be analysis for immunoglobulin gene rearrangements. Although we realize tttat it engenders controversy, we believe that the clinical a n d i m m u n o a r c h i t e c t u r a l data in cases 3 and ,t justify the suggestion tltat these infiltrates are reactive/aberrant rather than malignant. T h e e p i d e r m o t r o p i c , nonspongiotic, p r e d o m i nantly T-cell infiltrate in case 5 was of tlte histologic type that must be distinguished from "prentycotic" conditions and early mycosis fimgoides. Quantitative uhrastrnctural analysis in this case indicated a benign, reactive process. Ahhottgh the routine histologic features were similar to the "parapsoriasis en plaque fornt of inycosis fungoides,"',s9 tire clinical feattnes were not consistent with large-plaque parapsoriasis, tire lesion that ntay t r a n s f o r m into c u t a n e o u s l y m p l t o m a (in about 10 per cent of cases, according to Lambert and Everett40). In addition, similar histologic changes ltave been described in a case of cntaneous pseudolyntpltonta occurring in a tattoo. 35 Altltougit no abnorntality o f the Leu-3/Leu-2 helper to suppressor ratio was detected in case 5, we believe tlmt interpretation o f this ratio ntay be difficult in a case o f tltis type, for several reasons: 1) a ntalignant population of helper T cells may be associated with a reactive popuhttion of suppressor "-I"cells, 2~ and it ntay be difficult to distinguislt histologically between the two in i m m u n o p e r o x i d a s e - s t a i n c d f r o z e n sections because o f limited histologic detail; and 2) benign and reactive/hyperplastic c o n d i t i o n s ntay be associated with substantial a l t e r a t i o n s o f tire Lett-3/Lett-2 ratio. 7A9 Cell suspension studies using flow cytometry may be important in tire categorization and under509

s t a n d i n g o f these p r e d o m i n a n t l y T-cell infihrates. Prospective immtmologic analysis of many more cases of this type, including serial study of individual patients over time, p e r h a p s by combined i m m u n o a r chitectura], flow cytometric, and ultrastructural analyses, will be necessary before definitive conchtsions can be made. We believe that detection o f early cutaneous lymplloma is the crucial problem a n d suspect that large-plaque parapsoriasis (clinically) and the histologic features described identify a population at moderate risk for the development o f cutaneous lymphoma, analogous to the situation of patients with hepatic cirrhosis, who are at risk for the development of hepatocellular carcinoma. 4~ O u r findings do not resolve all of the diagnostic dilemmas that have been encountered in several of the difficult cases studied. T h e y do not suggest a "magic immunologic wand." T h e y do indicate, however, that the study of immunologic patterns in serial tissue sections with muhiple phcnotypic markers, i.e., immunoarclfitectural analysis, is a usefid tool for understanding the biology of cutaneons lynaphoproliferative disease, with eventual diagnostic utility likely. Imnatmoarchitectural analysis is likely to complen~ent the diagnostic utility o f quantitative tdtrastructural analysis a n d cell suspension imntunologic analysis. We speculate that the subtle immunologic aberrance found in some cases with no evidence Of monoclonality nmy prove to be usefnl for identifying a group at risk for malignant transformation and therefore requiring close follow-np evaluation. Acknowledgment. The authors thank Jack M. La)ton, MD, Chief of Pathology, University of Arizona Heahh Sciences Center, for his constant support. REFERENCES

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